Sphingomyelinases type-C (E.C. 3.1.4.12) are a group of phospholipases that catalyze the hydrolytic cleavage of sphingomyelin to ceramide and phosphocholine (Chatterjee, S. (1993) Adv. Lipid Res. 26:25–48). Native neutral sphingomyelinase (N-SMase) purified from human urine and cultured human kidney proximal tubular cell membranes has an apparent molecular weight of 92 kDa, neutral pH optima, is heat unstable, and is localized on the surface of various cells (Chatterjee, S. (1993) supra; Chatterjee, S. et al. (1989) J. Biol. Chem. 264:12,534–12,561; and Chatterjee, S. et al. (1991) Methods Enzymol., Phospholipase 197:540–547).
Cleavage of sphingomyelin and the products of this reaction have been implicated in multiple pathways, including apoptosis, cellular growth, differentiation, and inflammatory responsiveness. Multiple forms of SMases have been described on the basis of their optimal pH and intracellular localization, including lysosomal acidic SMase (A-SMase), cytosolic Zn2+-dependent acidic SMase, membrane-bound, magenesium-dependent neutral SMase (N-SMase), cytosolic magnesium-independent N-SMase, and alkaline SMase (Chatterjee, S. (1999) Chem Phy. Lipids 102, 79–9; Liu, B. and Hannun, Y. (1997) J. Biol. Chem. 272: 16281–16287; Martin, S. F. and Chatterjee, S. (2003) Methods Enzymol. (in press); Cordon-Cardo, C. and Kolesnick, R., (2001) Science 293–297).
N-SMase action has been shown to mediate signal transduction of vitamin D3, tumor necrosis factor-α (TNF-α), interferon-gamma, and nerve growth factor (Y. Hannun, J. Biol. Chem., 269:3,125–3,128 (1994); S. Chatterjee, J. Biol. Chem., 268:3,401–3,406 (1993); and S. Chatterjee, J. Biol. Chem., 269:879–882 (1994)) leading to cell differentiation in human leukemic (HL-60) cells and insulin signaling (P. Peraldi et al., J. Biol. Chem., 271:13018–13022 (1996)). Several investigators have identified apoptosis of smooth muscle cells (SMC) in atherosclerotic plaques. Even in early stages of atherosclerosis, apoptosis of SMC occurs (Hannun, 2002. FASEB J.). The loss of these cells due to apoptosis can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. It can be concluded that apoptosis in atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis (Hannun, 2002. FASEB J.).
In addition to the biological roles of sphingomyelin and ceramide in signal transduction pathways involving cell regulation, recent evidence suggests that sphingomyelinases may be involved in the mobilization of cell surface cholesterol, in cholesterol ester synthesis, and in induction of low density lipoprotein (LDL) receptor activity. See S. Chatterjee, Advances in Lipid Research, 26:25–48 (1993). Recent evidence also supports a possible role of ceramide (a product of N-SMase activity) in programmed cell death and/or “apoptosis” and activation of the gene for nuclear factor (NF)-κB. See A. Alessenko and S. Chatterjee, Mol. Cell. Biochem., 143:169–174 (1995). Sphingomyelinases are also believed to serve as a signal for various exogenous effectors such as antibiotics, drugs, and growth factors, which influence the normal physiology of cells.
It has been shown previously that high concentrations of Ox-LDL (100 μg/ml) can induce the death of aortic smooth muscle cells (ASMCs). A novel aspect of Ox-LDL mediated signal transduction to explain the phenomenon above was subsequently uncovered. In ASMCs, Ox-LDL stimulates the activity of N-SMase, an enzyme that cleaves sphingomyelin to ceramide and phosphocholine. The activity of N-SMase increased 5-fold within 5 minutes of incubation of cells with Ox-LDL, but not LDL, and then returned to baseline values after 30 minutes (Chatterjee, S. et al. (1999) J. Biol. Chem. 274:37407–37412). This was accompanied by marked apoptosis in cells incubated with Ox-LDL, as evidenced by (i) a DNA laddering, (ii) [3H]thymidine release, and (iii) fluorescence-assisted cell sorting analysis.
A number of specific disorders have been associated with N-SMase. For example, N-SMase has been reported to be associated with insulin resistant diabetes and obesity. See Speigel et al., J. Biol. Chem., 1996. N-SMase is also associated with malaria. The development of the malaria parasite plasmodium requires N-SMase. See Lauer et al., Proc. Nat. Acad. Sci. (USA), 1995. N-SMase also is involved in liver cell proliferation. See Alessenko, Chatterjee, Mol. Cell Biochem., 143:169–174 (1995).
Because of the involvement of N-SMase in a number of disorders, it would be desirable to have agents that are capable of downregulating N-SMase activity. However, such agents are currently not available.